This multi-disciplinary group of investigators has several years' experience working together designing and characterizing viral vector approaches to gene therapy of malignant brain tumors. A major focus has been producing and testing both non-replicative and replicative adenovirus (Ad) and conditionally replicative herpes simplex virus (HSV) vectors that express foreign gene products within infected tumor cells. These studies have been conducted at both the in vitro and in vivo levels to demonstrate proof-of-principle, safety and efficacy in experimental mouse models of intracranial gliomas. We have conducted Phase I and III clinical trials using retrovirus, Ad and HSV administered intratumorally in patients with malignant gliomas. In keeping with the translational theme of this SPORE application, this project seeks to design and deploy effective viral vector therapies of malignant glioma by utilizing rational combinations of foreign gene-viral vectors, oncolytic virus and irradiation., defined by additive, synergistic or antagonistic interactions determined for these various modalities. Aim 1 seeks to optimize the timing and dose of irradiation to achieve greater viral replication and spread and/or enhanced foreign gene expression in glioma cells and in intracranial experimental gliomas. In athymic nude mice. Aim 2 will develop and characterize both replicative HSV and replicative Ad that expression the pro-drug converting enzyme cytosine deaminase and optimize its use in intracranial preclinical models of malignant gliomas in combination with systemic 5-fluorocytosine. Other genetic constructs (uracil phosphoribosyl transferase) and drugs (dihydropyrimidine inhibitors) that facilitate appropriate 5-FU incorporation into host cell DNA synthesis pathways will also be tested to improve the therapeutic effect. Further, the radiation sensitization properties of certain pro-drugs products (5-FU) will be characterized to achieve a greater anti-glioma effect. Aim 3 will combine findings in Aims 1 and 2 to design and test strategies that rationally combine intratumoral viral vector injection, systemic pro-drug administration and low dose external beam irradiation to achieve the most effective and safe anti-glioma therapy (ies). Aim 4 will translate our findings in preclinical models for brain tumor therapy into pilot, Phase I and Phase II clinical trials in patients with malignant gliomas.